Benzidine testing

Benzidine testing of bone surface has been shown to yield a strong positive reaction in recent bone samples (Facchini and Pettener 1977 Knight and Lauder 1969). The intensity of the reaction will decrease with time, and a negative result indicates an older bone sample. As the test relies on the presence of blood, the results are susceptible to the rapid loss of blood remnants due to environmental conditions. However, if these factors are considered, the benzidine reaction can supply a useful indicator for identifying skeletal remains of potential forensic interest. 

Another test dependent on the presence of blood is luminol testing. As with the benzidine test, the intensity will decrease as the age of the bone increases due to the loss of hemoglobin proteins (Introna, Di Vella, and Cam-pobasso 1999). A negative result is indicative of older bone samples. Luminol testing is perhaps the most recent development in chemical techniques used to date skeletal remains. The method provides a good correlation between the levels of intensity of the chemiluminescence with the difference in time since death of the bones (ibid.). 

Ammonium molybdate-benzidine test (DANGER THE REAGENT IS CARCINOGENIC.) Silicates react with molybdates in acid solution to form the complex silicomolybdic acid H4[SiMo12O40]. The ammonium salt, unlike the analogous phosphoric acid and arsenic acid compounds, is soluble in water and acids to give a yellow solution. The test depends upon the reaction between silicomolybdic acid and benzidine in acetic acid solution whereby molybdenum blue and a blue quinonoid oxidation compound of benzidine are produced. 

Ammonium molybdate-benzidine test (DANGER THE REAGENT IS... 

The colors produced from ketoses, pentoses, and uronic acids in the presence of phenols and acids as well as other reagents often are enough different from those formed from aldohexoses so that they may be used for the classification of unknown materials. The ketoses, pentoses, and uronic acids usually form colored products under conditions milder than those required for the aldohexoses. Tauber s benzidine test for pentoses and... 

The reductive pyrolysis of organic compounds in the presence of metals leads to the formation of the metal cyanide, which can be detected as Prussian blue (Fe4[Fe(CN)g]3) or by the copper(ii) acetate-benzidine test . Several metals and salts have been recommended for the fusion of the organic compound, e.g. potassium , sodium , magnesium mixed with potassium carbonate , zinc mixed with potassium carbonate , and a mixture of dextrose with sodium carbonate . When the compound contains sulphur the metal thiocyanate is also produced, which can be detected by ferric chloride . ... 

This method of detecting cerium in the presence of other rare earths (especially zirconium and thorium salts) gives better limiting proportions than the benzidine test (see page 175). The comparative figures for 10 y cerium are ... 

The test is not directly applicable in the presence of manganese, cobalt, copper, silver, thallium, or chromates because they, too, oxidize benzidine. In these instances it is advisable to precipitate cerous fluoride from the neutral or slighly acid test solution with hydrofluoric acid, then to produce Ce ii hydroxide by spotting with alkali, and apply the benzidine test. 

This method for the detection of germanium is only decisive in the absence of certain other materials. Apart from compounds which reduce molybdates directly—e.g., Sn, Fe, As, and Se —arsenic acid, phosphoric acid and silicic acid should not be present, as they also form heteropoly molybdic acids, which enter into the same redox reaction with benzidine. The germanium can, however, be distilled out of hydrochloric acid solution (3.5-4.0 N) as Ge v chloride. The molybdate-benzidine test is then carried out with the distillate. 

Tests (1) Benzidine Test.— Add about 10 drops of a fresh, strong solution of benzidine in glacial acetic acid to 1 ml. of 3 per cent. (10 vol. Og per 100 vol.) hydrogen peroxide. Add 1 ml. of urine, and mix well. If blood be present the mixture turns dark blue. 

A mixture of 280 g. (1.52 moles) of commercial benzidine and 880 cc. (10.23 moles) of concentrated hydrochloric acid (sp. gr. 1.182) is placed in a 5-I. round-bottomed flask and wanned on a steam bath for one to two hours, with occasional shaking, to form the dihydrochloride. The flask is then equipped with a mechanical stirrer and a dropping funnel, and cooled, with stirring, to — ro° in an ice-salt bath. When this temperature has been reached, the benzidine dihydrochloride is tetrazotized over a period of two hours with a solution of 232 g. (3.19 moles) of 95 per cent sodium nitrite in 800 cc. of water, until a faint test for nitrous acid with starch-iodide paper is obtained after twenty minutes. During this reaction, the temperature is kept below —5 °. 

It is important for acid-catalysed reactions to determine whether the reaction is specifically catalysed by hydrogen ions or whether general acid catalysis takes place. Specific acid catalysis has been conclusively demonstrated for the benzidine rearrangement by three different sorts of kinetic experiments. In the first, it has been shown41 by the standard test for general acid catalysis (by measuring the rate of reaction in a buffered solution at constant pH over a range of concentration... 

Tauber test (analy chem) A color test for identification of pentose sugars the sugars produce a cherry-red color when heated with a solution of benzidine in glacial acetic acid. tau-bor, test ... 

No cases of bladder tumors were found in an epidemiology study of 259 workers exposed to dry and semi-dry 3,3 -dichlorobenzidine base and hydrochloride. Cytological analyses of the urine (Papanicolaou tests) were negative. Workers were exposed to an average of less than 16 years each to 3,3 -dichloro-benzidine, which means that an adequate exposure dmation and/or the latent period following exposm-e may not have been reached for tumor expression (MacIntyre 1975). 

Ocular Effects. No studies were located regarding ocular effects in humans after exposure to 3,3 -dichlorobenzidine by any route. No adverse effects on the eye were noted when dichlorobenzidine (isomer unspecified, free base) was directly placed in the conjunctival sac of the eye of rabbits (Gerarde and Gerarde 1974). However, 0.1 mL 3,3 -dichlorobenzidine dihydrochloride (dihydro salt of 3,3 -dichloro-benzidine) in a 20% com oil suspension produced erythema, pus, and comeal opacity, giving a 76% score in the Draize test within an hour when placed in the conjunctival sac of the eye of the rabbit (Gerarde and Gerarde 1974). Apparently, the irritant effects of hydrochloric acid Ifom the salt-compoimd contributed... 

Gamer RC, Walpole AL, Rose FL. 1975. Testing of some benzidine analogues for microsomal activation to bacterial mutagens. Cancer Lett l(l) 39-42. 

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